A single amino acid substitution in the R3 domain of GLABRA1 leads to inhibition of trichome formation in Arabidopsis without affecting its interaction with GLABRA3

GLABRA1 (GL1) is an R2R3 MYB transcription factor that regulates trichome formation in Arabidopsis by interacting with the bHLH transcription factor GLABRA3 (GL3) or ENHANCER OF GL3 (EGL3). The conserved [D/E]Lx2 [R/K]x3Lx6Lx3R amino acid signature in the R3 domain of MYB proteins has been shown to be required for the interaction of MYBs with R/B-like bHLH transcription factors. By using genetic and molecular analyses, we show that the glabrous phenotype in the nph4-1 mutant is caused by a single nucleotide mutation in the GL1 gene, generating a Ser to Phe substitution (S92F) in the conserved [D/E]Lx2[R/K]x3Lx6Lx3R amino acid signature of GL1. Activation of the integrated GL2p:GUS reporter gene in protoplasts by cotransfection of GL1 and GL3 or EGL3 was abolished by this GL1-S92F substitution. However, GL1-S92F interacted successfully with GL3 or EGL3 in protoplast transfection assays. Unlike VPGL1GL3, the fusion protein VPGL1-S92FGL3 failed to activate the integrated GL2p:GUS reporter gene in transfected protoplasts. These results suggested that the S92 in the conserved [D/E]Lx2 [R/K]x3Lx6Lx3R amino acid signature of GL1 is not essential for the interaction of GL1 and GL3, but may play a role in the binding of GL1 to the promoters of its target genes. The R2R3 MYB transcription factor GL1 is a key regulator of trichome formation in Arabidopsis. The conserved [D/E]Lx2[R/K]x3Lx6Lx3R amino acid signature in the R3 domain is required for the interaction of MYBs with R/B-like bHLH transcription factors. S92F amino acid substantiation in the conserved [D/E]Lx2[R/K]x3Lx6Lx3R signature in GL1 lead to loss-of-function mutation of GL1. However, our results indicate that Ser92 residue is not required for the interaction of GL1 with bHLH transcription factor GL3 or EGL3, but may required for binding of GL1 to its target genes.